The microbial conversion of sustainable lignocellulosic biomass into biofuels could provide a source of fully renewable transportation fuels (1). Generating these fuels from abundant feedstocks such as lignocellulose and cellulosic waste avoids many of the problems associated with current grain-based biofuels, provided the feedstock is responsibly grown and harvested (2). While early efforts toward achieving economical biofuel production have typically focused on improving yields of ethanol made from fermentation of plant sugars (3), recent advances in metabolic engineering have enabled microbial production of fuels that are compatible with existing engines and fuel distribution infrastructure (4, 5). Many of these advances have been made possible by the unparalleled genetic and metabolic tractability of the model bacterium Escherichia coli (6, 7). E. coli has been engineered to biosynthesize perhaps the most chemically diverse range of chemicals of any organism, including hydrogen (8), higher alcohols (9, 10), fatty-acid based chemicals (11), and terpenes (12, 13). Extensive knowledge of E. coli physiology will continue to aid improvements in titers beyond those achieved in proof-of-concept stages toward levels required for a commercial-scale biofuel production process.
Unfortunately, several challenges must be overcome before lignocellulose can be considered an economically competitive feedstock for biofuel production. One of the more significant challenges is the need for large quantities of glycoside hydrolase (GH) enzymes to efficiently convert lignocellulose into fermentable sugars. These enzymes are typically generated in a dedicated process that incurs substantial capital and material expense and represent the second highest contribution to raw material cost after the feedstock itself (1, 14). An alternative approach, known as consolidated bioprocessing, could potentially avoid the costs of a dedicated enzyme generation step by performing it in a combined process that includes biomass hydrolysis and fuel production (FIG. 1A) (15, 16). This can be achieved by incorporating both biomass-degrading and biofuel-producing capabilities into a single organism through genetic engineering. Several microorganisms have been engineered to ferment model cellulosic and hemicellulosic substrates directly into ethanol or other fuels (reviewed in refs. 15 and 17). For example, the yeast Saccharomyces cerevisiae (18) and the bacterium Klebsiella oxytoca (19) have been modified to convert phosphoric acid swollen cellulose (PASC) directly to ethanol without the addition of exogenous cellulase. However, PASC and similar model substrates are typically prepared using techniques that are neither suitable for actual plant biomass nor feasible on a large scale (20). Furthermore, no biofuel with the combustion properties of petrochemical fuels, which could be used directly in existing infrastructure, has been generated directly from unrefined lignocellulosic biomass.
A cellulolytic strain of E. coli capable of growth on plant biomass would be a first step toward producing many varieties of advanced biofuels at lowered cost. One obstacle to engineering E. coli for consumption of lignocellulose is the organism's inferior capacity for protein export, which renders it unable to secrete cellulases in quantities required for industrial-scale lignocellulose hydrolysis. Various techniques, developed over decades of research, can be applied to generate secreted yields from E. coli of 0.5-0.8 g protein/L (21). Unfortunately, these concentrations are still too low for an industrial process, which are most efficient around levels of 20 mg cellulase/g solids and 200 g/L solids loading (22) [although recent work (23) has demonstrated that removal of soluble hydrolase inhibitors may substantially reduce the enzyme loading required]. To further engineer a cellulolytic E. coli strain for use in consolidated bioproces sing, biofuel production pathways must also be introduced and expressed at levels that yield high titers while not overburdening the cell. The integration of engineered cellulolytic capabilities together with pathways for advanced biofuel production into a single organism may present an insurmountable metabolic burden for E. coli, or indeed any microbe, without appropriate regulation.